Controlling the expression of heterologous genes in Bdellovibrio bacteriovorus using synthetic biology strategies

Abstract Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that preys upon Gram‐negative bacteria. It has been proposed to be applied as a “living antibiotic” in several fields such as agriculture or even medicine, since it is able to prey upon bacterial pathogens. Its interesting lifestyle makes this bacterium very attractive as a microbial chassis for co‐culture systems including two partners. A limitation to this goal is the scarcity of suitable synthetic biology tools for predator domestication. To fill this gap, we have firstly adapted the hierarchical assembly cloning technique Golden Standard (GS) to make it compatible with B. bacteriovorus HD100. The chromosomal integration of the Tn7 transposon's mobile element, in conjunction with the application of the GS technique, has allowed the systematic characterization of a repertoire of constitutive and inducible promoters, facilitating the control of the expression of heterologous genes in this bacterium. PJExD/EliR proved to be an exceptional promoter/regulator system in B. bacteriovorus HD100 when precise regulation is essential, while the synthetic promoter PBG37 showed a constitutive high expression. These genetic tools represent a step forward in the conversion of B. bacteriovorus into an amenable strain for microbial biotechnology approaches.


Supplementary Tables
List of strains used throughout this work.

Strain
Genotype Reference

E. coli DH5α
Cloning host; F-λ -endAI hsdRJ7 (rk -, mk + ) supE44 thi- This work vector containing PJ23106-BCD2-mRFP1-rnpB_T1 This work This work vector containing PBG25-BCD2-mRFP1-rnpB_T1 This work vector containing PBG37-BCD2-mRFP1-rnpB_T1 This work This work This work This work This work This work This work This work               Two successive pre-cultures were grown supplementing with 10 μg • mL -1 Gm using E. coli BL21(DE3) Gm R as prey at OD600 1 in Hepes + .The second pre-culture was measured by flow cytometry (0 h) and was used to inoculate fresh prey supplementing with the indicated Gm concentrations.After 24 h, the predated co-cultures were analyzed again by flow cytometry (24 h).In the histograms are represented the percentage of positive (+) and negative (-) events over the total events.The name of the plasmids is represented by the promoter of each construction, the backbone for all the GS constructions is pSEVA6519[g2].

Figure S2 .
Figure S2.Analysis of the attTn7 site in B. bacteriovorus HD100.A) Localization of the attTn7 site in the genome of B. bacteriovorus HD100 and scheme of the transposase recognition site.The chromosomal integration mediated by the transposon Tn7 comprises four proteins TnsA, TnsB, TnsC, and TnsD.TnsD is the protein that binds to the 3' region of the glmS gene.TnsC is a protein that joins the transposase (TnsAB) and the TnsD protein (Peters, 2014).The machinery for the mobile element transposition can be supplied in a suicide plasmid (pTnS-1), avoiding the mobile element dispersion and off-target events (Choi et al., 2005).B) Multiple alignment of experimentally validated TnsD binding regions.The consensus logo marks critical nucleotides for TnsD binding with a red asterisk.Nucleotides whose mutation has a lower effect on TnsD binding are marked with a yellow asterisk (Mitra et al., 2010).Logo was generated with WebLogo (https://weblogo.berkeley.edu/logo.cgi),using default colors.Alignment was done with ClustalO 1.2 with the R package 'msa' (Bodenhofer et al., 2015).

Figure S3 .
Figure S3.GS constructions made for the inducible expression of msfGFP in B. bacteriovorus HD100.The msfGFP CDS was assembled with the Pm, PrhaBAD, PJExD, Pb, and PBAD promoters, keeping constant the other parts, i.e., the translational coupler BCD2 and the rnpB_T1 terminator, yielding five Level 1 constructions.These Level 1 constructions were mixed with the Level 1 plasmids carrying the regulator of each inducible system and the Level 2 destination vector, pTn7-19[g2], yielding five Level 2 plasmids with the MsfGFP constructions in the Tn7 mobile element.

Figure S4 .
Figure S4.Comparison of the RpoD σ factors of E. coli K-12 and B. bacteriovorus HD100.Domains of each RpoD are schematically represented, with their amino acid conservation represented above (in yellow bars, with increased darkness indicating lower conservation).The sequence of the subdomains involved in the recognition of -35 and -10 promoter's boxes (the subdomain 4.2 and 2.4, respectively) are compared below.

Figure S5 .
Figure S5.Phase-contrast and fluorescence microscopy images of mRFP1 harboring plasmids B. bacteriovorus HD100 strains.Pictures of 24-hours of co-cultures in Hepes+ supplemented with 5 μg • mL -1 of Gm and E. coli BL21(DE3) Gm R as prey.

Figure S6 .
Figure S6.Stability of plasmid vs Tn7 integration in B. bacteriovorus HD100.B. bacteriovorus Gm R viable cells in successive co-cultures with and without antibiotic pressure.A) Data obtained from the strain carrying the conferring resistance plasmid, pSEVA6519[g2]-ncTU.B) Data obtained from the strain with the Gm R cassette in the chromosome, Bb08-ncTU.The strains were grown in E. coli BL21(DE3) Gm R suspensions at OD600 1 in Hepes + .Error bars represent the SD of two biological replicates.Two-way ANOVA with Bonferroni post-test was applied to compare results within each propagation level (* means P < 0.1 and ** P < 0.05).

Figure S7 .
Figure S7.Evolution of fluorescent populations of plasmids bearing B. bacteriovorus HD100 strains.Two successive pre-cultures were grown supplementing with 10 μg • mL -1 Gm using E. coli BL21(DE3) Gm R as prey at OD600 1 in Hepes + .The second pre-culture was measured by flow cytometry (0 h) and was used to inoculate fresh prey supplementing with the indicated Gm concentrations.After 24 h, the predated co-cultures were analyzed again by flow cytometry (24 h).In the histograms are represented the percentage of positive (+) and negative (-) events over the total events.The name of the plasmids is represented by the promoter of each construction, the backbone for all the GS constructions is pSEVA6519[g2].

Figure S8 .
Figure S8.Single-cell fluorescence analysis of B. bacteriovorus strains with inducible promoters chromosomal integrated.The strains were grown in E. coli BL21(DE3) Gm R suspensions at OD600 1, in Hepes + supplemented with the indicated concentrations of the respective inducers.At 24 h, the predated co-cultures were analyzed by flow cytometry.B. bacteriovorus HD100 was used as the negative control.A) Stacked histogram corresponding to the Bb-PJExD-G strain.B) Stacked histogram corresponding to the Bb-Pb-G strain.

Table S6 . Constructed Golden Standard Level 0 parts
†Restriction enzyme used to make construct; Sp, streptomycin; Sm, spectinomycin.* GS reaction was done with 100 ng of annealed primers instead of PCR product.

Table S8 . Evaluation of the synthetic promoter library in B. bacteriovorus HD100
Relative activity of each promoter, taking the highest expressing promoter as reference.Calculated with the median of the fluorescent population obtained analyzing by flow cytometry (FC) 24-hours predated co-cultures in flasks.b Events • mL -1 : Total events measured by flow cytometry of a 24-h co-culture grown in flask that served as inoculum for the microplate experiment.c Initial PFUs of the microplate experiment were measured following the double-layer method.d Total events measured by flow cytometry of the predated co-cultures grown in a microplate reader.e Maximal death rate (h -1 ): Maximal rate of the OD600 absorbance decrease, i.e., the absorbance of the prey.f Time in h when the decrease in the OD600 is maximal.g Time in h when the increase of the fluorescence is maximal.

Table S9 . Assessment of the impact of increasing concentrations of Crystal Violet (CV) on the Bb08-PJExD-G strain in microplate experiment
Initial PFUs of the microplate experiment measured following the double-layer method.b Total events measured by flow cytometry (FC) of the predated co-cultures grown in a microplate reader.c Maximal death rate (h -1 ): Maximal rate of the OD600 absorbance decrease, i.e., the absorbance of the prey.d Time in h when the decrease in the OD600 is maximal.e Time in h when the increase of the fluorescence is maximal.